Control of cellular senescence by CPEB.

Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA.

Abstract

Cytoplasmic polyadenylation element-binding protein (CPEB) is a sequence-specific RNA-binding protein that promotes polyadenylation-induced translation. While a CPEB knockout (KO) mouse is sterile but overtly normal, embryo fibroblasts derived from this mouse (MEFs) do not enter senescence in culture as do wild-type MEFs, but instead are immortal. Exogenous CPEB restores senescence in the KO MEFs and also induces precocious senescence in wild-type MEFs. CPEB cannot stimulate senescence in MEFs lacking the tumor suppressors p53, p19ARF, or p16(INK4A); however, the mRNAs encoding these proteins are unlikely targets of CPEB since their expression is the same in wild-type and KO MEFs. Conversely, Ras cannot induce senescence in MEFs lacking CPEB, suggesting that it may lie upstream of CPEB. One target of CPEB regulation is myc mRNA, whose unregulated translation in the KO MEFs may cause them to bypass senescence. Thus, CPEB appears to act as a translational repressor protein to control myc translation and resulting cellular senescence.

CPEB controls senescence in MEFs. (A, left) A Western blot of wild-type (WT) and CPEB KO MEFs probed for CPEB and tubulin. (Right) The rate of population doublings of four CPEB KO MEF lines and, for comparison, one wild-type line and one CPEB heterozygous line. The MEFs were cultured according to a 3T3 protocol. (B) Wild-type and CPEB KO MEFs were infected with a retrovirus expressing HA-tagged CPEB, or an empty control vector, at passage 2 and then fixed at passage 4 (wild type) or passage 11 (KO) and stained for β-galactosidase activity. Also shown is a Western blot demonstrating the expression of HA-tagged CPEB in wild-type and KO MEFs. (C) MEFs lacking CPEB, p19ARF, p16INK/p19ARF, or p53 were infected with the CPEB-encoding virus or an empty vector at passage 4 and examined by phase contrast microscopy at passage 11. (D) MEFs derived from wild-type, CPEB KO, and p19ARF KO animals were infected at passage 2 with a retrovirus expressing Ki-Ras or an empty vector, selected by puromycin at passage 3, and examined by phase contrast microscopy at passage 5.

CPEB KO MEFs have features consistent with partial transformation. (A) Wild-type (WT) and CPEB KO MEFs were examined by phase contrast microscopy and immunostained for α-tubulin at passages 3 and 25 (P3 and P25) to assess cell morphology. The cells were also stained with DAPI to visualize nuclei. (B) Wild-type and CPEB KO MEFs were cultured for up to 5 d in medium containing 1% or 10% serum and analyzed for cell number by staining with crystal violet (average ± SD, n = 3). Some of the MEFs were also infected with a retrovirus expressing Ki-Ras and were similarly examined. (C) Wild-type and CPEB KO MEFs, some of which were infected with a Ki-Ras-containing virus or an empty virus vector as before, were grown on soft agar for 2 wk. Small foci were estimated to have ~100 cells, while large foci were estimated to have ~500–1000 cells.

Tumors derived from wild-type (WT) and CPEB KO MEFs transformed with Ki-Ras. Immortalized cells derived from wild-type MEFs grown according to a 3T3 protocol as well as CPEB KO MEFs were infected with a retrovirus expressing Ki-Ras. The cells were injected subcutaneously into athymic (nude) mice (five mice each, two injections per mouse near each hind limb). All Ki-Ras-containing cells formed tumors irrespective of genotype (10/10 WT, 10/10 CPEB KO). (A) Representative pictures of the tumors at the sixth week after injection; note the hematoma on the tumors derived from the CPEB KO MEFs (cf. those denoted by arrows). All 10 tumors derived from the Ras-transformed KO cells had hematomas. (B) Tumor growth. (C) Final tumor and mouse weight 6 wk after injection. (D) The tumors shown in A were excised, sectioned, and stained with hematoxylin and eosin. Representative sections are shown. The arrows denote cells with enlarged cytoplasms and displaced nuclei.

Expression levels of proteins involved in the cell cycle and cell senescence. Wild-type (WT) and CPEB KO MEFs were collected over passages 2–8 and examined by Western blot ting for the indicated proteins. Tubulin served as the loading control and as a standard for film exposures among blots. Be cause the p53 antibody reacted strongly with two proteins from wild-type and CPEB KO MEFs, extracts from p53 KO and parallel wild-type MEFs were also analyzed. The faster migrating protein was absent from the p53 KO MEFs, demonstrating that it is p53. (NS) Nonspecific band.

RNA binding but not phosphorylation is necessary for CPEB rescue of senescence. The CPEB KO MEFs were infected with virus expressing no CPEB (Vec), wild-type CPEB (WT), CPEB lacking the RRMs (ΔRRM), CPEB lacking the zinc finger (ΔZF), or CPEB with T171A/S177A mutations that prevent cytoplasmic polyadenylation. The cells were selected and counted on days 3 and 6. The Western blot shows the relative levels of the virally expressed proteins.

Translational regulation of myc mRNA by CPEB. (A) MEFs were infected with Flag-and HA-tagged CPEB and subsequently subjected to RNP coimmunoprecipitation sequentially with antibody directed against each epitope. The RNA was extracted from the final precipitation and subjected to RT–PCR for the denoted RNAs. In addition to the immunoprecipitated RNP complexes, total RNA from wild-type (WT) and KO MEFs was serially diluted and analyzed for RNA levels by RT–PCR. (Right) The relative input levels of the RNAs by RT–PCR from wild-type, CPEB KO, and CPEB KO MEFs that were infected with virus expressing CPEB. (B) 32P myc 3′ UTR was mixed with recombinant CPEB and, in some cases, with CPE-containing (CPE+) or CPE-lacking (CPE−) RNA. The mixture was UV-irradiated, RNase-digested, and analyzed by SDS-PAGE and PhosphorImaging. (C) MEFs infected with the same plasmids as in A were mixed with CPE-containing (CPE+) or CPE-lacking (CPE−) RNAs prior to HA immunoprecipitation and RT–PCR for myc RNA. (D) Wild-type and CPEB KO MEFs at passage 4 were centrifuged through sucrose gradients and fractionated, and the myc and actin RNAs in each fraction were determined by quantitative real-time PCR. The histogram depicts the amount of myc RNA relative to actin RNA in each fraction. The UV scans of the gradients (absorbance at 254 nm) are depicted at the top. (E) Schematic diagram of Renilla luciferase RNA appended with either the myc 3′ UTR (the CPEs and the polyadenylation hexanucleotide AAUAAA are shown) or a 3′ UTR derived from vector sequences but containing the AAUAAA. Each of these in vitro synthesized RNAs was mixed with RNA encoding firefly (FF) luciferase appended with a 3′ UTR vector sequence, and transfected into wild-type and CPEB KO MEFs. The FF luciferase construct served as an internal control for transfection efficiency. The amount of Renilla luciferase in each of the MEFs was then determined 5 h after transfection and expressed as the fold change in CPEB KO versus wild-type MEFs. The average of three experiments (±SD) is shown.

Myc RNA translation by CPEB controls senescence. (A) At passage 2, wild-type (WT) and CPEB KO MEFs were transfected with a retrovirus expressing the myc-coding region appended with its own 3′ UTR or a 3′ UTR derived from vector sequences. Some MEFs were infected with a virus containing only the vector. At passage 7, cell number was assessed by microscopy and by counting with a hemocytometer. Some of the cells were also used for Western blotting for myc and tubulin, and for RT–PCR of the myc ORF and 3′ UTR. In addition, the myc 3′ UTR was quantified by real-time PCR (histogram). The levels of this RNA were made relative to actin RNA, which was also quantified by real-time PCR. (B, top panel) KO MEFs were infected with shRNAs for myc or GFP at passage 2 and puromycin-selected, and a Western analysis for myc protein was performed at passage 5. The cells were also examined for population doubling (middle panel) and phase contrast microscopy (bottom panel).